Methods of decreasing background on a spatial array

ABSTRACT

Provided herein are methods of determining a location of a target analyte in a non-permeabilized biological sample and methods of reducing background binding of an analyte on an array.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 62/960,573, filed on Jan. 13,2020, the entire contents of which are hereby incorporated by reference.

BACKGROUND

Cells within a tissue have differences in cell morphology and/orfunction due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, signaling, and cross-talk with other cells inthe tissue.

Spatial heterogeneity has been previously studied using techniques thattypically provide data for a handful of analytes in the context ofintact tissue or a portion of a tissue (e.g., tissue section), orprovide significant analyte data from individual, single cells, butfails to provide information regarding the position of the single cellsfrom the originating biological sample (e.g., tissue).

SUMMARY

This application provides for an improvement between signal to noise(e.g., background analyte binding) during performance of any of themethods described herein for determining a location of a target analytein a biological sample.

Provided herein are methods for decreasing background binding of atarget nucleic acid on an array that include: (a) disposing a biologicalsample onto an array; in some embodiments, the array has a first areacovered by the biological sample and a second area not covered by thebiological sample; in some embodiments, the array comprises a pluralityof capture probes; in some embodiments, a capture probe of the pluralityof capture probes comprises a spatial barcode and a capture domain; (b)contacting the second area of the array with a solution comprising adiffusion-restricted nuclease, (c) removing the diffusion-restrictednuclease from the second area of the array; and (d) permeabilizing thebiological sample, such that the capture domain binds to the targetnucleic acid in the first area, thereby decreasing the backgroundbinding of a target nucleic acid on the array.

In some embodiments of any of the methods described herein, the methodsfurther include determining (i) all or a portion of the sequence of thespatial barcode, or a complement thereof, and (ii) all or a portion ofthe sequence of the target nucleic acid, or a complement thereof, andusing the sequences of (i) and (ii) to determine the location of thetarget nucleic acid in the biological sample.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease degrades single-stranded nucleic acids. Insome embodiments of any of the methods described herein, thediffusion-restricted nuclease degrades double-stranded nucleic acids.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is covalently linked to a bead, aparticle, or a polymer. In some embodiments of any of the methodsdescribed herein, the polymer is a polyethylene glycol.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is an endonuclease or an exonuclease. Insome embodiments of any of the methods described herein, the exonucleaseis a 3′ to 5′ exonuclease. In some embodiments of any of the methodsdescribed herein, the exonuclease is a DNAse.

In some embodiments of any of the methods described herein, the removingcomprises washing.

In some embodiments of any of the methods described herein, the arraycomprises a slide. In some embodiments of any of the methods describedherein, the array is a bead array.

In some embodiments of any of the methods described herein, thedetermining comprises sequencing (i) all or a portion of the sequence ofthe spatial barcode, or a complement thereof, and (ii) all or a portionof the sequence of the target nucleic acid, or a complement thereof. Insome embodiments of any of the methods described herein, the sequencingis high throughput sequencing.

In some embodiments of any of the methods described herein, thedetermining comprises extending the capture probe using the targetnucleic acid as the template.

In some embodiments of any of the methods described herein, steps (a)and (b) are performed at substantially the same time.

Also provided herein are methods for determining a location of a targetnucleic acid in a biological sample disposed onto an array, where thearray has a first area covered by the biological sample and a secondarea not covered by the biological sample, where the array comprises aplurality of capture probes, where a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain, whereinthe methods include: (a) contacting the second area of the array with asolution comprising a diffusion-restricted nuclease; (b) removing thediffusion-restricted nuclease from the second area of the array; (c)permeabilizing the biological sample, such that the capture domain bindsto the target nucleic acid; and (d) determining (i) all or a portion ofthe sequence of the spatial barcode, or a complement thereof, and (ii)all or a portion of the sequence of the target nucleic acid, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target nucleic acid in the biological sample.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease degrades single-stranded nucleic acids. Insome embodiments of any of the method described herein, thediffusion-restricted nuclease degrades double-stranded nucleic acids.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is covalently linked to a bead, aparticle, or a polymer. In some embodiments of the methods describedherein, the polymer is a polyethylene glycol.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is an endonuclease or an exonuclease. Insome embodiments of any of the methods described herein, the exonucleaseis a 3′ to 5′ exonuclease. In some embodiments of any of the methodsdescribed herein, the exonuclease is a DNAse.

In some embodiments of any of the methods described herein, the removingin step (b) comprises washing. In some embodiments of any of the methodsdescribed herein, the array comprises a slide. In some embodiments ofany of the methods described herein, the array is a bead array.

In some embodiments of any of the methods described herein, thedetermining in step (d) comprises sequencing (i) all or a portion of thesequence of the spatial barcode, or a complement thereof, and (ii) allor a portion of the sequence of the target nucleic acid, or a complementthereof. In some embodiments of any of the methods described herein, thesequencing is high throughput sequencing.

In some embodiments of any of the methods described herein, thedetermining in step (d) includes extending the capture probe using thetarget nucleic acid as the template.

In some embodiments of any of the methods described herein, thebiological sample is a tissue section. In some embodiments of any of themethods described herein, the tissue section is from a fresh frozentissue section.

Also provided herein are methods for determining a location of a targetanalyte in a biological sample that include: (a) contacting a pluralityof analyte capture agents to the biological sample; in some embodiments,an analyte capture agent of the plurality of analyte capture agentscomprises an analyte binding moiety barcode, an analyte capturesequence, and an analyte binding moiety that binds specifically to thetarget analyte; (b) disposing the biological sample onto an array; insome embodiments, the array has a first area covered by the biologicalsample and a second area not covered by the biological sample; in someembodiments, the array comprises a plurality of capture probes; in someembodiments, a capture probe of the plurality comprises a spatialbarcode and a capture domain that binds specifically to the analytecapture sequence; (c) contacting the second area of the array with asolution comprising a diffusion-restricted nuclease; (d) removing thediffusion-restricted nuclease from the second area of the array; and (e)determining (i) all or a portion of the sequence of the spatial barcode,or a complement thereof, and (ii) all or a portion of the sequence ofthe analyte binding moiety barcode, or a complement thereof, and usingthe sequences of (i) and (ii) to determine the location of the targetanalyte in the biological sample.

In some embodiments of any of the methods described herein, step (a) isperformed before step (b). In some embodiments of any of the methodsdescribed herein, step (b) is performed before step (a).

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease degrades single-stranded nucleic acids. Insome embodiments of any of the methods described herein, thediffusion-restricted nuclease degrades double-stranded nucleic acids.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is covalently linked to a bead, aparticle, or a polymer. In some embodiments of any of the methodsdescribed herein, the polymer is a polyethylene glycol.

In some embodiments of any of the methods described herein, thediffusion-restricted nuclease is an endonuclease or an exonuclease. Insome embodiments of any of the methods described herein, the exonucleaseis a 3′ to 5′ exonuclease. In some embodiments of any of the methodsdescribed herein, the exonuclease is a DNAse.

In some embodiments of any of the methods described herein, the removingin step (d) comprises washing.

In some embodiments of any of the methods described herein, the arraycomprises a slide. In some embodiments of any of the methods describedherein, the array is a bead array.

In some embodiments of any of the methods described herein, thedetermining in step (e) comprises sequencing (i) all or a portion of thesequence of the spatial barcode, or a complement thereof, and (ii) allor a portion of the sequence of the analyte binding moiety barcode, or acomplement thereof. In some embodiments of any of the methods describedherein, the sequencing is high throughput sequencing.

In some embodiments of any of the methods described herein, thedetermining in step (e) comprises extending the capture probe using theanalyte binding moiety barcode as the template.

In some embodiments of any of the methods described herein, steps (a)and (b) are performed at substantially the same time.

In some embodiments of any of the methods described herein, the methodsfurther include after step (d): permeabilizing the biological sampledisposed on the array, such that the analyte binding moiety binds to thetarget analyte and the capture domain binds to the analyte capturesequence.

In some embodiments of any of the methods described herein, thebiological sample is a tissue section. In some embodiments of any of themethods described herein, the tissue section is a fresh, frozen tissuesection.

Also provided herein are kits that include an array comprising aplurality of capture probes, where a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain; and adiffusion-restricted nuclease.

Also provided herein are kits that include a plurality of analytecapture agents, where an analyte capture agent of the plurality ofanalyte capture agents comprises an analyte binding moiety barcode, ananalyte capture sequence, and an analyte binding moiety; an arraycomprising a plurality of capture probes, where a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain; and a diffusion-restricted nuclease.

As used herein, the term “non-permeabilized biological sample” means abiological sample that has not been exposed to one or morepermeabilizing agents (e.g., any of the exemplary permeabilizing agentsdescribed herein), or has not been subjected to a permeabilizationmethod (e.g., any of the exemplary permeabilization methods describedherein).

As used herein, the term “diffusion-restricted nuclease” means anuclease that has is covalently and/or non-covalently attached to anagent that results in a decrease in the rate of diffusion of thenuclease as compared to the rate of diffusion of the same nuclease notcovalently or non-covalently attached to the agent. In some embodiments,a diffusion-restricted nuclease is not able to significantly passthrough an intact plasma membrane of a non-permeabilized mammalian cell.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 shows an example of background signal resulting from RNA in anarea of an array where a brain tissue sample had not been placed.

FIG. 2 shows an example of background signal resulting from protein inan area of an array where a spleen tissue sample had not been placed.

DETAILED DESCRIPTION

The methods described herein provide for an improvement between signalto background during performance of any of the methods described hereinfor determining a location of a target analyte in a biological sample.

Provided herein are methods for determining a location of a targetnucleic acid in a biological sample disposed onto an array, where thearray has a first area covered by the biological sample and a secondarea not covered by the biological sample, where the array comprises aplurality of capture probes, where a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain, wherethe methods include: (a) contacting the second area of the array with asolution comprising a diffusion-restricted nuclease; (b) removing thediffusion-restricted nuclease from the second area of the array; (c)permeabilizing the biological sample, such that the capture domain bindsto the target nucleic acid; and (d) determining (i) all or a portion ofthe sequence of the spatial barcode, or a complement thereof, and (ii)all or a portion of the sequence of the target nucleic acid, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target nucleic acid in the biological sample.

Provided herein are methods for determining a location of a targetanalyte in a biological sample that include: (a) contacting a pluralityof analyte capture agents to the biological sample, where an analytecapture agent of the plurality of analyte capture agents comprises ananalyte binding moiety barcode, an analyte capture sequence, and ananalyte binding moiety that binds specifically to the target analyte;(b) disposing the biological sample onto an array, where the array has afirst area covered by the biological sample and a second area notcovered by the biological sample, where the array comprises a pluralityof capture probes, where a capture probe of the plurality comprises aspatial barcode and a capture domain that binds to the analyte capturesequence; (c) contacting the second area of the array with a solutioncomprising a diffusion-restricted nuclease; (d) removing thediffusion-restricted nuclease from the second area of the array; and (e)determining (i) all or a portion of the sequence of the spatial barcode,or a complement thereof, and (ii) all or a portion of the sequence ofthe analyte binding moiety barcode, or a complement thereof, and usingthe sequences of (i) and (ii) to determine the location of the targetanalyte in the biological sample.

Provided herein are methods for decreasing background binding of atarget nucleic acid on an array that include: (a) disposing a biologicalsample onto an array, where the array has a first area covered by thebiological sample and a second area not covered by the biologicalsample, where the array comprises a plurality of capture probes, where acapture probe of the plurality of capture probes comprises a spatialbarcode and a capture domain; (b) contacting the second area of thearray with a solution comprising a diffusion-restricted nuclease, (c)removing the diffusion-restricted nuclease from the second area of thearray; and (d) permeabilizing the biological sample, such that thecapture domain binds to the target nucleic acid in the first area,thereby decreasing the background binding of a target nucleic acid onthe array.

Provided herein are kits that include: an array comprising a pluralityof capture probes, where a capture probe of the plurality of captureprobes comprises a spatial barcode and a capture domain; and adiffusion-restricted nuclease.

Provided herein are kits that include: a plurality of analyte captureagents, where an analyte capture agent of the plurality of analytecapture agents comprises an analyte binding moiety barcode, an analytecapture sequence, and an analyte binding moiety; an array comprising aplurality of capture probes, where a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain; and adiffusion-restricted nuclease.

Some embodiments of any of the methods described herein can provide forat least a 0.1-fold improvement, at least a 0.5-fold improvement, atleast a 0.8-fold improvement, at least a 1-fold improvement, at least a1.5-fold improvement, at least a 2-fold improvement, at least a 2.5-foldimprovement, at least a 3-fold improvement, at least a 3.5-foldimprovement, at least a 4-fold improvement, at least a 4.5-foldimprovement, at least a 5-fold improvement, at least a 5.5-foldimprovement, at least a 6-fold improvement, at least a 6.5-foldimprovement, at least a 7-fold improvement, at least a 7.5-foldimprovement, at least a 8-fold improvement, at least a 8.5-foldimprovement, at least a 9-fold improvement, at least a 9.5-foldimprovement, at least 10-fold improvement, at least a 15-foldimprovement, at least a 20-fold improvement, at least 30-foldimprovement, at least 50-fold improvement, or at least 100-foldimprovement in the signal to noise ratio as compared to a similar methodperformed without the use of a diffusion-restricted nuclease. See, e.g.,FIG. 1 and FIG. 2.

Some embodiments of any of the methods described herein can provide forabout a 0.1-fold improvement to about a 100-fold improvement, about a0.1-fold improvement to about a 50-fold improvement, about a 0.1-foldimprovement to about a 30-fold improvement, about a 0.1-fold improvementto about a 20-fold improvement, about a 0.1-fold improvement to about a15-fold improvement, about a 0.1-fold improvement to about a 10-foldimprovement, about a 0.1-fold improvement to about a 8-fold improvement,about a 0.1-fold improvement to about a 6-fold improvement, about a0.1-fold improvement to about a 4-fold improvement, about a 0.1-foldimprovement to about a 2-fold improvement, about a 0.1-fold improvementto about a 1-fold improvement, about a 0.1-fold improvement to about a0.8-fold improvement, about a 0.1-fold improvement to about a 0.6-foldimprovement, about a 0.1-fold improvement to about a 0.4-foldimprovement, about a 0.1-fold improvement to about a 0.2-foldimprovement, about a 0.2-fold improvement to about a 100-foldimprovement, about a 0.2-fold improvement to about a 50-foldimprovement, about a 0.2-fold improvement to about a 30-foldimprovement, about a 0.2-fold improvement to about a 20-foldimprovement, about a 0.2-fold improvement to about a 15-foldimprovement, about a 0.2-fold improvement to about a 10-foldimprovement, about a 0.2-fold improvement to about a 8-fold improvement,about a 0.2-fold improvement to about a 6-fold improvement, about a0.2-fold improvement to about a 4-fold improvement, about a 0.2-foldimprovement to about a 2-fold improvement, about a 0.2-fold improvementto about a 1-fold improvement, about a 0.2-fold improvement to about a0.8-fold improvement, about a 0.2-fold improvement to about a 0.6-foldimprovement, about a 0.2-fold improvement to about a 0.4-foldimprovement, about a 0.4-fold improvement to about a 100-foldimprovement, about a 0.4-fold improvement to about a 50-foldimprovement, about a 0.4-fold improvement to about a 30-foldimprovement, about a 0.4-fold improvement to about a 20-foldimprovement, about a 0.4-fold improvement to about a 15-foldimprovement, about a 0.4-fold improvement to about a 10-foldimprovement, about a 0.4-fold improvement to about a 8-fold improvement,about a 0.4-fold improvement to about a 6-fold improvement, about a0.4-fold improvement to about a 4-fold improvement, about a 0.4-foldimprovement to about a 2-fold improvement, about a 0.4-fold improvementto about a 1-fold improvement, about a 0.4-fold improvement to about a0.8-fold improvement, about a 0.4-fold improvement to about a 0.6-foldimprovement, about a 0.6-fold improvement to about a 100-foldimprovement, about a 0.6-fold improvement to about a 50-foldimprovement, about a 0.6-fold improvement to about a 30-foldimprovement, about a 0.6-fold improvement to about a 20-foldimprovement, about a 0.6-fold improvement to about a 15-foldimprovement, about a 0.6-fold improvement to about a 10-foldimprovement, about a 0.6-fold improvement to about a 8-fold improvement,about a 0.6-fold improvement to about a 6-fold improvement, about a0.6-fold improvement to about a 4-fold improvement, about a 0.6-foldimprovement to about a 2-fold improvement, about a 0.6-fold improvementto about a 1-fold improvement, about a 0.6-fold improvement to about a0.8-fold improvement, about a 0.8-fold improvement to about a 100-foldimprovement, about a 0.8-fold improvement to about a 50-foldimprovement, about a 0.8-fold improvement to about a 30-foldimprovement, about a 0.8-fold improvement to about a 20-foldimprovement, about a 0.8-fold improvement to about a 15-foldimprovement, about a 0.8-fold improvement to about a 10-foldimprovement, about a 0.8-fold improvement to about a 8-fold improvement,about a 0.8-fold improvement to about a 6-fold improvement, about a0.8-fold improvement to about a 4-fold improvement, about a 0.8-foldimprovement to about a 2-fold improvement, about a 0.8-fold improvementto about a 1-fold improvement, about a 1-fold improvement to about a100-fold improvement, about a 1-fold improvement to about a 50-foldimprovement, about a 1-fold improvement to about a 30-fold improvement,about a 1-fold improvement to about a 20-fold improvement, about a1-fold improvement to about a 15-fold improvement, about a 1-foldimprovement to about a 10-fold improvement, about a 1-fold improvementto about a 8-fold improvement, about a 1-fold improvement to about a6-fold improvement, about a 1-fold improvement to about a 4-foldimprovement, about a 1-fold improvement to about a 2-fold improvement,about a 2-fold improvement to about a 100-fold improvement, about a2-fold improvement to about a 50-fold improvement, about a 2-foldimprovement to about a 30-fold improvement, about a 2-fold improvementto about a 20-fold improvement, about a 2-fold improvement to about a15-fold improvement, about a 2-fold improvement to about a 10-foldimprovement, about a 2-fold improvement to about a 8-fold improvement,about a 2-fold improvement to about a 6-fold improvement, about a 2-foldimprovement to about a 4-fold improvement, about a 4-fold improvement toabout a 100-fold improvement, about a 4-fold improvement to about a50-fold improvement, about a 4-fold improvement to about a 30-foldimprovement, about a 4-fold improvement to about a 20-fold improvement,about a 4-fold improvement to about a 15-fold improvement, about a4-fold improvement to about a 10-fold improvement, about a 4-foldimprovement to about a 8-fold improvement, about a 4-fold improvement toabout a 6-fold improvement, about a 6-fold improvement to about a100-fold improvement, about a 6-fold improvement to about a 50-foldimprovement, about a 6-fold improvement to about a 30-fold improvement,about a 6-fold improvement to about a 20-fold improvement, about a6-fold improvement to about a 15-fold improvement, about a 6-foldimprovement to about a 10-fold improvement, about a 6-fold improvementto about a 8-fold improvement, about a 8-fold improvement to about a100-fold improvement, about a 8-fold improvement to about a 50-foldimprovement, about a 8-fold improvement to about a 30-fold improvement,about a 8-fold improvement to about a 20-fold improvement, about a8-fold improvement to about a 15-fold improvement, about a 8-foldimprovement to about a 10-fold improvement, about a 10-fold improvementto about a 100-fold improvement, about a 10-fold improvement to about a50-fold improvement, about a 10-fold improvement to about a 30-foldimprovement, about a 10-fold improvement to about a 20-fold improvement,about a 10-fold improvement to about a 15-fold improvement, about a15-fold improvement to about a 100-fold improvement, about a 15-foldimprovement to about a 50-fold improvement, about a 15-fold improvementto about a 30-fold improvement, about a 15-fold improvement to about a20-fold improvement, about a 20-fold improvement to about a 100-foldimprovement, about a 20-fold improvement to about a 50-fold improvement,about a 20-fold improvement to about a 30-fold improvement, about a30-fold improvement to about a 100-fold improvement, about a 30-foldimprovement to about a 50-fold improvement, or about a 50-foldimprovement to about 100-fold improvement, in the signal to noise ratioas compared to a similar method performed without the use of adiffusion-restricted nuclease.

Some embodiments of any of the methods described herein can provide forat least a 0.1-fold improvement, at least a 0.5-fold improvement, atleast a 1-fold improvement, at least a 1.5-fold improvement, at least a2-fold improvement, at least a 2.5-fold improvement, at least a 3-foldimprovement, at least a 3.5-fold improvement, at least a 4-foldimprovement, at least a 4.5-fold improvement, at least a 5-foldimprovement, at least a 5.5-fold improvement, at least a 6-foldimprovement, at least a 6.5-fold improvement, at least a 7-foldimprovement, at least a 7.5-fold improvement, at least a 8-foldimprovement, at least a 8.5-fold improvement, at least a 9-foldimprovement, at least a 9.5-fold improvement, at least 10-foldimprovement, at least a 15-fold improvement, at least a 20-foldimprovement, at least 30-fold improvement, at least 50-fold improvement,at least 100-fold improvement in the sequencing yield per spot ascompared to a similar method performed without the use of adiffusion-restricted nuclease.

Some embodiments of any of the methods described herein can provide forabout a 0.1-fold improvement to about a 100-fold improvement, about a0.1-fold improvement to about a 50-fold improvement, about a 0.1-foldimprovement to about a 30-fold improvement, about a 0.1-fold improvementto about a 20-fold improvement, about a 0.1-fold improvement to about a15-fold improvement, about a 0.1-fold improvement to about a 10-foldimprovement, about a 0.1-fold improvement to about a 8-fold improvement,about a 0.1-fold improvement to about a 6-fold improvement, about a0.1-fold improvement to about a 4-fold improvement, about a 0.1-foldimprovement to about a 2-fold improvement, about a 0.1-fold improvementto about a 1-fold improvement, about a 0.1-fold improvement to about a0.5-fold improvement, about a 0.1-fold improvement to about a 0.2-foldimprovement, about a 0.2-fold improvement to about a 100-foldimprovement, about a 0.2-fold improvement to about a 50-foldimprovement, about a 0.2-fold improvement to about a 30-foldimprovement, about a 0.2-fold improvement to about a 20-foldimprovement, about a 0.2-fold improvement to about a 15-foldimprovement, about a 0.2-fold improvement to about a 10-foldimprovement, about a 0.2-fold improvement to about a 8-fold improvement,about a 0.2-fold improvement to about a 6-fold improvement, about a0.2-fold improvement to about a 4-fold improvement, about a 0.2-foldimprovement to about a 2-fold improvement, about a 0.2-fold improvementto about a 1-fold improvement, about a 0.2-fold improvement to about a0.5-fold improvement, about a 0.5-fold improvement to about a 100-foldimprovement, about a 0.5-fold improvement to about a 50-foldimprovement, about a 0.5-fold improvement to about a 30-foldimprovement, about a 0.5-fold improvement to about a 20-foldimprovement, about a 0.5-fold improvement to about a 15-foldimprovement, about a 0.5-fold improvement to about a 10-foldimprovement, about a 0.5-fold improvement to about a 8-fold improvement,about a 0.5-fold improvement to about a 6-fold improvement, about a0.5-fold improvement to about a 4-fold improvement, about a 0.5-foldimprovement to about a 2-fold improvement, about a 0.5-fold improvementto about a 1-fold improvement, about a 1-fold improvement to about a100-fold improvement, about a 1-fold improvement to about a 50-foldimprovement, about a 1-fold improvement to about a 30-fold improvement,about a 1-fold improvement to about a 20-fold improvement, about a1-fold improvement to about a 15-fold improvement, about a 1-foldimprovement to about a 10-fold improvement, about a 1-fold improvementto about a 8-fold improvement, about a 1-fold improvement to about a6-fold improvement, about a 1-fold improvement to about a 4-foldimprovement, about a 1-fold improvement to about a 2-fold improvement,about a 2-fold improvement to about a 100-fold improvement, about a2-fold improvement to about a 50-fold improvement, about a 2-foldimprovement to about a 30-fold improvement, about a 2-fold improvementto about a 20-fold improvement, about a 2-fold improvement to about a15-fold improvement, about a 2-fold improvement to about a 10-foldimprovement, about a 2-fold improvement to about a 8-fold improvement,about a 2-fold improvement to about a 6-fold improvement, about a 2-foldimprovement to about a 4-fold improvement, about a 4-fold improvement toabout a 100-fold improvement, about a 4-fold improvement to about a50-fold improvement, about a 4-fold improvement to about a 30-foldimprovement, about a 4-fold improvement to about a 20-fold improvement,about a 4-fold improvement to about a 15-fold improvement, about a4-fold improvement to about a 10-fold improvement, about a 4-foldimprovement to about a 8-fold improvement, about a 4-fold improvement toabout a 6-fold improvement, about a 6-fold improvement to about a100-fold improvement, about a 6-fold improvement to about a 50-foldimprovement, about a 6-fold improvement to about a 30-fold improvement,about a 6-fold improvement to about a 20-fold improvement, about a6-fold improvement to about a 15-fold improvement, about a 6-foldimprovement to about a 10-fold improvement, about a 6-fold improvementto about a 8-fold improvement, about a 8-fold improvement to about a100-fold improvement, about a 8-fold improvement to about a 50-foldimprovement, about a 8-fold improvement to about a 30-fold improvement,about a 8-fold improvement to about a 20-fold improvement, about a8-fold improvement to about a 15-fold improvement, about a 8-foldimprovement to about a 10-fold improvement, about a 10-fold improvementto about a 100-fold improvement, about a 10-fold improvement to about a50-fold improvement, about a 10-fold improvement to about a 30-foldimprovement, about a 10-fold improvement to about a 20-fold improvement,about a 10-fold improvement to about a 15-fold improvement, about a15-fold improvement to about a 100-fold improvement, about a 15-foldimprovement to about a 50-fold improvement, about a 15-fold improvementto about a 30-fold improvement, about a 15-fold improvement to about a20-fold improvement, about a 20-fold improvement to about a 100-foldimprovement, about a 20-fold improvement to about a 50-fold improvement,about a 20-fold improvement to about a 30-fold improvement, about a30-fold improvement to about a 100-fold improvement, about a 30-foldimprovement to about a 50-fold improvement, or about a 50-foldimprovement to about a 100-fold improvement, in the sequencing yield perspot as compared to a similar method performed without the use of adiffusion-restricted nuclease

Additional non-limiting aspects of these methods are described hereinand can be used in any combination.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain).

A “spatial barcode” is a contiguous nucleic acid segment or two or morenon-contiguous nucleic acid segments that function as a label oridentifier that conveys or is capable of conveying spatial information.In some embodiments, a capture probe includes a spatial barcode thatpossesses a spatial aspect, where the barcode is associated with aparticular location within an array or a particular location on asubstrate.

The “capture domain” can be an oligonucleotide, a polypeptide, a smallmolecule, or any combination thereof, that binds specifically to adesired analyte. In some embodiments, a capture domain can be used tocapture or detect a desired analyte.

In some embodiments, a capture probe can include a cleavage domainand/or a functional domain (e.g., a primer-binding site, such as fornext-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g.,subsections (i)-(vi)) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Generation of capture probes can beachieved by any appropriate method, including those described in Section(II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent.

The term “analyte binding moiety” refers to a molecule or moiety capableof binding to a macromolecular constituent (e.g., an analyte, e.g., abiological analyte). In some embodiments of any of the spatial profilingmethods described herein, the analyte binding moiety of the analytecapture agent that binds to a biological analyte can include, but is notlimited to, an antibody, or an epitope binding fragment thereof, a cellsurface receptor binding molecule, a receptor ligand, a small molecule,a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptorengager, a B-cell receptor engager, a pro-body, an aptamer, a monobody,an affimer, a darpin, and a protein scaffold, or any combinationthereof. The analyte binding moiety can bind to the macromolecularconstituent (e.g., analyte) with high affinity and/or with highspecificity. The analyte binding moiety can include a nucleotidesequence (e.g., an oligonucleotide), which can correspond to at least aportion or an entirety of the analyte binding moiety. The analytebinding moiety can include a polypeptide and/or an aptamer (e.g., apolypeptide and/or an aptamer that binds to a specific target molecule,e.g., an analyte). The analyte binding moiety can include an antibody orantibody fragment (e.g., an antigen-binding fragment) that binds to aspecific analyte (e.g., a polypeptide).

Additional description of analyte capture agents can be found in Section(II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. PatentApplication Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationsproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein. In some cases, the systems described herein can detect(e.g., register an image) the biological sample on the array. Exemplarymethods to detect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

I. First and Second Areas

Some embodiments of any of the methods described herein includedisposing a biological sample (e.g., a non-permeabilized biologicalsample) onto an array (e.g., any of the exemplary arrays describedherein), where the array has a first area covered by thenon-permeabilized biological sample and a second area not covered by thenon-permeabilized biological sample.

The first area can represent a portion of the array that is covered bythe biological sample, e.g., at least 1%, at least 2%, at least 4%, atleast 6%, at least 8%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%, of the total area of the array covered by the biologicalsample.

In some examples, the first area can represent a portion of the arraythat is covered by the biological sample, e.g., about 1% to about 100%,about 1% to about 95%, about 1% to about 90%, about 1% to about 85%,about 1% to about 80%, about 1% to about 75%, about 1% to about 70%,about 1% to about 65%, about 1% to about 60%, about 1% to about 55%,about 1% to about 50%, about 1% to about 45%, about 1% to about 40%,about 1% to about 35%, about 1% to about 30%, about 1% to about 35%,about 1% to about 20%, about 1% to about 15%, about 1% to about 10%,about 10% to about 100%, about 10% to about 90%, about 10% to about 80%,about 10% to about 70%, about 10% to about 60%, about 10% to about 50%,about 10% to about 40%, about 10% to about 30%, about 10% to about 20%,about 20% to about 100%, about 20% to about 90%, about 20% to about 80%,about 20% to about 70%, about 20% to about 60%, about 20% to about 50%,about 20% to about 40%, about 20% to about 30%, about 30% to about 100%,about 30% to about 90%, about 30% to about 80%, about 30% to about 70%,about 30% to about 60%, about 30% to about 50%, about 30% to about 40%,about 40% to about 100%, about 40% to about 90%, about 40% to about 80%,about 40% to about 70%, about 40% to about 60%, about 40% to about 50%,about 50% to about 100%, about 50% to about 90%, about 50% to about 80%,about 50% to about 70%, about 50% to about 60%, about 60% to about 100%,about 60% to about 90%, about 60% to about 80%, about 60% to about 70%,about 70% to about 100%, about 70% to about 90%, about 70% to about 80%,about 80% to about 100%, about 80% to about 90%, or about 90% to about100%, of the total area of the array covered by the biological sample.

The second area can represent a portion of the array that is not coveredby the biological sample, e.g., at least 1%, at least 2%, at least 4%,at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%, of the total area of the array not covered by the biologicalsample. In some examples, the second area can represent a portion of thearray that is not covered by the biological sample, e.g., about 1% toabout 100%, about 1% to about 95%, about 1% to about 90%, about 1% toabout 85%, about 1% to about 80%, about 1% to about 75%, about 1% toabout 70%, about 1% to about 65%, about 1% to about 60%, about 1% toabout 55%, about 1% to about 50%, about 1% to about 45%, about 1% toabout 40%, about 1% to about 35%, about 1% to about 30%, about 1% toabout 35%, about 1% to about 20%, about 1% to about 15%, about 1% toabout 10%, about 10% to about 100%, about 10% to about 90%, about 10% toabout 80%, about 10% to about 70%, about 10% to about 60%, about 10% toabout 50%, about 10% to about 40%, about 10% to about 30%, about 10% toabout 20%, about 20% to about 100%, about 20% to about 90%, about 20% toabout 80%, about 20% to about 70%, about 20% to about 60%, about 20% toabout 50%, about 20% to about 40%, about 20% to about 30%, about 30% toabout 100%, about 30% to about 90%, about 30% to about 80%, about 30% toabout 70%, about 30% to about 60%, about 30% to about 50%, about 30% toabout 40%, about 40% to about 100%, about 40% to about 90%, about 40% toabout 80%, about 40% to about 70%, about 40% to about 60%, about 40% toabout 50%, about 50% to about 100%, about 50% to about 90%, about 50% toabout 80%, about 50% to about 70%, about 50% to about 60%, about 60% toabout 100%, about 60% to about 90%, about 60% to about 80%, about 60% toabout 70%, about 70% to about 100%, about 70% to about 90%, about 70% toabout 80%, about 80% to about 100%, about 80% to about 90%, or about 90%to about 100%, of the total area of the array not covered by thebiological sample.

II. Diffusion-Restricted Nucleases

In some embodiments, the nuclease present is a diffusion-restricted, anenzyme capable of cleaving the phosphodiester bonds between nucleotidesof nucleic acids (e.g. RNA and/or DNA). In some embodiments, thenuclease degrades single-strand nucleic acids (e.g., single-stranded DNAand/or single-stranded RNA). In some embodiments, the nuclease degradesdouble-strand nucleic acids (e.g., double-stranded DNA, double-strandedRNA, or a double-stranded DNA/RNA molecule). In some embodiments, thenuclease is an endonuclease. In some embodiment, the nuclease is anexonuclease. In some embodiments, the nuclease is a deoxyribonuclease(DNAse). In some embodiments, the nuclease is a ribonuclease (RNAse). Insome embodiments, the nuclease is a 3′ to 5′ exonuclease. In someembodiments, the nuclease is a DNAse I (e.g., bovine pancreatic DNAseI). In some embodiments, the nuclease is a DNAse II. In someembodiments, the nuclease is a wild-type DNAse. In some embodiments, thenuclease comprises an amino acid sequence that is at least 70%, 75%,80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence ofa wild-type DNase. In some embodiments, the nuclease comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,about 30, about 40, or about 50 amino acid substitutions compared to theamino acid sequence of a wild-type DNAse. In some embodiments, thenuclease is a recombinant nuclease. In some embodiments, the nuclease isexpressed in a host bacterial strain (e.g., E. coli) or a host cell(e.g., insect cell). In some embodiments, the nuclease comprisesadditional amino acid sequences that can facilitate recombinant nucleaseexpression and purification (e.g., poly-histidine tag or GST tag). Insome embodiments, the recombinant nuclease is encoded by a nucleic acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to the nucleic acid sequence encoding a wild-type DNase. Insome embodiments, the recombinant nuclease is encoded by a nucleic acidsequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, or about 100 mutations (e.g., pointmutations) compared to the nucleic acid sequence encoding a wild-typeDNAse.

In some embodiments, the diffusion-restricted nuclease is covalentlyconjugated or non-covalently attached to a bead. For example, a bead nothaving a capture probe attached but having a similar shape, dimension,material, size, coating, appearance, and/or physical properties, ascompared to any of the other beads described herein.

In some embodiments, the diffusion-restricted nuclease is covalentlyconjugated or non-covalently attached to a particle. In someembodiments, the particle can be rigid. In some embodiments, theparticle can be flexible and/or compressible. A particle can generallybe of any suitable shape. Examples of particle shapes include, but arenot limited to, spherical, non-spherical, oval, oblong, amorphous,circular, cylindrical, cuboidal, hexagonal, and variations thereof.Particles can be of uniform size or heterogeneous size.

In some embodiments, the particle can have a diameter or maximumdimension no larger than 100 μm (e.g., no larger than 95 μm, 90 μm, 85μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm,8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm).

In some embodiments, a plurality of particles has an average diameter nolarger than 100 μm. In some embodiments, a plurality of particles has anaverage diameter or maximum dimension no larger than 95 μm, 90 μm, 85μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm,8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm.

In some embodiments, the volume of the particle can be at least about 1μm³, e.g., at least 1 μm³, 2 μm³, 3 μm³, 4 μm³, 5 μm³, 6 μm³, 7 μm³, 8μm³, 9 μm³, 10 μm³, 12 μm³, 14 μm³, 16 μm³, 18 μm³, 20 μm³, 25 μm³, 30μm³, 35 μm³, 40 μm³, 45 μm³, 50 μm³, 55 μm³, 60 μm³, 65 μm³, 70 μm³, 75μm³, 80 μm³, 85 μm³, 90 μm³, 95 μm³, 100 μm³, 125 μm³, 150 μm³, 175 μm³,200 μm³, 250 μm³, 300 μm³, 350 μm³, 400 μm³, 450 μm³, μm³, 500 μm³, 550μm³, 600 μm³, 650 μm³, 700 μm³, 750 μm³, 800 μm³, 850 μm³, 900 μm³, 950μm³, 1000 μm³, 1200 μm³, 1400 μm³, 1600 μm³, 1800 μm³, 2000 μm³, 2200μm³, 2400 μm³, 2600 μm³, 2800 μm³, 3000 μm³, or greater.

In some embodiments, the particle can have a volume of between about 1μm³ and 100 μm³, such as between about 1 μm³ and 10 μm³, between about10 μm³ and 50 μm³, or between about 50 μm³ and 100 μm³. In someembodiments, the particle can include a volume of between about 100 μm³and 1000 μm³, such as between about 100 μm³ and 500 μm³ or between about500 μm³ and 1000 μm³. In some embodiments, the particle can include avolume between about 1000 μm³ and 3000 μm³, such as between about 1000μm³ and 2000 μm³ or between about 2000 μm³ and 3000 μm³. In someembodiments, the particle can include a volume between about 1 μm³ and3000 μm³, such as between about 1 μm³ and 2000 μm³, between about 1 μm³and 1000 μm³, between about 1 μm³ and 500 μm³, or between about 1 μm³and 250 μm³.

The particle can include two or more cross-section(s) that can be thesame or different. In some embodiments, the particle can have a firstcross-section that is different from a second cross-section. Theparticle can have a first cross-section that is at least about 0.0001micrometer, at least 0.001 micrometer, at least 0.01 micrometer, atleast 0.1 micrometer, or at least 1 micrometer. In some embodiments, theparticle can include a cross-section (e.g., a first cross-section) of atleast about 1 micrometer (μm), at least about 2 μm, at least about 3 μm,at least about 4 μm, at least about 5 μm, at least about 6 μm, at leastabout 7 μm, at least about 8 μm, at least about 9 μm, at least about 10μm, at least about 11 μm, at least about 12 μm, at least about 13 μm, atleast about 14 μm, at least about 15 μm, at least about 16 μm, at leastabout 17 μm, at least about 18 μm, at least about 19 μm, at least about20 μm, or at least about 25 μm. In some embodiments, the particle caninclude a cross-section (e.g., a first cross-section) of about 0.01 μmto about 25 μm, about 0.01 μm to about 20 μm, about 0.01 μm to about 18μm, about 0.01 μm to about 16 μm, about 0.01 μm to about 14 μm, about0.01 μm to about 12 μm, about 0.01 μm to about 10 μm, about 0.01 μm toabout 8 μm, about 0.01 μm to about 6 μm, about 0.01 μm to about 4 μm,about 0.01 μm to about 2 μm, about 0.01 μm to about 1 μm, about 0.01 μmto about 0.5 μm, about 0.01 μm to about 0.1 μm, about 0.01 μm to about0.05 μm, about 0.05 μm and 25 μm, about 0.05 μm to about 20 μm, about0.05 μm to about 18 μm, about 0.05 μm to about 16 μm, about 0.05 μm toabout 14 μm, about 0.05 μm to about 12 μm, about 0.05 μm to about 10 μm,about 0.05 μm to about 8 μm, about 0.05 μm to about 6 μm, about 0.05 μmto about 4 μm, about 0.05 μm to about 2 μm, about 0.05 μm to about 1 μm,about 0.05 μm to about 0.5 μm, about 0.05 μm to about 0.1 μm, about 0.1μm and 25 μm, about 0.1 μm to about 20 μm, about 0.1 μm to about 18 μm,about 0.1 μm to about 16 μm, about 0.1 μm to about 14 μm, about 0.1 μmto about 12 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 8 μm,about 0.1 μm to about 6 μm, about 0.1 μm to about 4 μm, about 0.1 μm toabout 2 μm, about 0.1 μm to about 1 μm, about 0.1 μm to about 0.5 μm,about 0.5 μm and 25 μm, about 0.5 μm to about 20 μm, about 0.5 μm toabout 18 μm, about 0.5 μm to about 16 μm, about 0.5 μm to about 14 μm,about 0.5 μm to about 12 μm, about 0.5 μm to about 10 μm, about 0.5 μmto about 8 μm, about 0.5 μm to about 6 μm, about 0.5 μm to about 4 μm,about 0.5 μm to about 2 μm, about 0.5 μm to about 1 μm, about 1 μm and25 μm, about 1 μm to about 20 μm, about 1 μm to about 18 μm, about 1 μmto about 16 μm, about 1 μm to about 14 μm, about 1 μm to about 12 μm,about 1 μm to about 10 μm, about 1 μm to about 8 μm, about 1 μm to about6 μm, about 1 μm to about 4 μm, about 1 μm to about 2 μm, about 2 μm and25 μm, about 2 μm to about 20 μm, about 2 μm to about 18 μm, about 2 μmto about 16 μm, about 2 μm to about 14 μm, about 2 μm to about 12 μm,about 2 μm to about 10 μm, about 2 μm to about 8 μm, about 2 μm to about6 μm, about 2 μm to about 4 μm, about 4 μm and 25 μm, about 4 μm toabout 20 μm, about 4 μm to about 18 μm, about 4 μm to about 16 μm, about4 μm to about 14 μm, about 4 μm to about 12 μm, about 4 μm to about 10μm, about 4 μm to about 8 μm, about 4 μm to about 6 μm, about 6 μm and25 μm, about 6 μm to about 20 μm, about 6 μm to about 18 μm, about 6 μmto about 16 μm, about 6 μm to about 14 μm, about 6 μm to about 12 μm,about 6 μm to about 10 μm, about 6 μm to about 8 μm, about 8 μm and 25μm, about 8 μm to about 20 μm, about 8 μm to about 18 μm, about 8 μm toabout 16 μm, about 8 μm to about 14 μm, about 8 μm to about 12 μm, about8 μm to about 10 μm, about 10 μm and 25 μm, about 10 μm to about 20 μm,about 10 μm to about 18 μm, about 10 μm to about 16 μm, about 10 μm toabout 14 μm, about 10 μm to about 12 μm, about 12 μm and 25 μm, about 12μm to about 20 μm, about 12 μm to about 18 μm, about 12 μm to about 16μm, about 12 μm to about 14 μm, about 14 μm and 25 μm, about 14 μm toabout 20 μm, about 14 μm to about 18 μm, about 14 μm to about 16 μm,about 16 μm and 25 μm, about 16 μm to about 20 μm, about 16 μm to about18 μm, about 18 μm and 25 μm, about 18 μm to about 20 μm, or about 20 μmto about 25 μm.

For example, the particle can include a cross-section (e.g., a firstcross-section) of about 1 μm to 100 μm. In some embodiments, theparticle can have a second cross-section that is at least about 1 μm.For example, the particle can include a second cross-section of at leastabout 1 μm, at least about 2 μm, at least about 3 μm, at least about 4μm, at least about 5 μm, at least about 6 μm, at least about 7 μm, atleast about 8 μm, at least about 9 μm, at least about 10 μm, at leastabout 11 μm, at least about 12 μm, at least about 13 μm, at least about14 μm, at least about 15 μm, at least about 16 μm, at least about 17 μm,at least about 18 μm, at least about 19 μm, at least about 20 μm, atleast about 25 μm, at least about 30 μm, at least about 35 μm, at leastabout 40 μm, at least about 45 μm, at least about 50 μm, at least about55 μm, at least about 60 μm, at least about 65 μm, at least about 70 μm,at least about 75 μm, at least about 80 μm, at least about 85 μm, atleast about 90 μm, at least about 100 μm, at least about 120 μm, atleast about 140 μm, at least about 160 μm, at least about 180 μm, atleast about 200 μm, at least about 250 μm, at least about 300 μm, atleast about 350 μm, at least about 400 μm, at least about 450 μm, or atleast about 500 μm. In some embodiments, the particle can include asecond cross-section of about 1 μm to about 500 μm, about 1 μm to about450 μm, about 1 μm to about 400 μm, about 1 μm to about 350 μm, about 1μm to about 300 μm, about 1 μm to about 250 μm, about 1 μm to about 200μm, about 1 μm to about 150 μm, about 1 μm to about 100 μm, about 1 μmto about 80 μm, about 1 μm to about 60 μm, about 1 μm to about 40 μm,about 1 μm to about 20 μm, about 1 μm to about 15 μm, about 1 μm toabout 10 μm, about 1 μm to about 8 μm, about 1 μm to about 6 μm, about 1μm to about 4 μm, about 1 μm to about 2 μm, about 2 μm to about 500 μm,about 2 μm to about 450 μm, about 2 μm to about 400 μm, about 2 μm toabout 350 μm, about 2 μm to about 300 μm, about 2 μm to about 250 μm,about 2 μm to about 200 μm, about 2 μm to about 150 μm, about 2 μm toabout 100 μm, about 2 μm to about 80 μm, about 2 μm to about 60 μm,about 2 μm to about 40 μm, about 2 μm to about 20 μm, about 2 μm toabout 15 μm, about 2 μm to about 10 μm, about 2 μm to about 8 μm, about2 μm to about 6 μm, about 2 μm to about 4 μm, about 4 μm to about 500μm, about 4 μm to about 450 μm, about 4 μm to about 400 μm, about 4 μmto about 350 μm, about 4 μm to about 300 μm, about 4 μm to about 250 μm,about 4 μm to about 200 μm, about 4 μm to about 150 μm, about 4 μm toabout 100 μm, about 4 μm to about 80 μm, about 4 μm to about 60 μm,about 4 μm to about 40 μm, about 4 μm to about 20 μm, about 4 μm toabout 15 μm, about 4 μm to about 10 μm, about 4 μm to about 8 μm, about4 μm to about 6 μm, about 6 μm to about 500 μm, about 6 μm to about 450μm, about 6 μm to about 400 μm, about 6 μm to about 350 μm, about 6 μmto about 300 μm, about 6 μm to about 250 μm, about 6 μm to about 200 μm,about 6 μm to about 150 μm, about 6 μm to about 100 μm, about 6 μm toabout 80 μm, about 6 μm to about 60 μm, about 6 μm to about 40 μm, about6 μm to about 20 μm, about 6 μm to about 15 μm, about 6 μm to about 10μm, about 6 μm to about 8 μm, about 8 μm to about 500 μm, about 8 μm toabout 450 μm, about 8 μm to about 400 μm, about 8 μm to about 350 μm,about 8 μm to about 300 μm, about 8 μm to about 250 μm, about 8 μm toabout 200 μm, about 8 μm to about 150 μm, about 8 μm to about 100 μm,about 8 μm to about 80 μm, about 8 μm to about 60 μm, about 8 μm toabout 40 μm, about 8 μm to about 20 μm, about 8 μm to about 15 μm, about8 μm to about 10 μm, about 10 μm to about 500 μm, about 10 μm to about450 μm, about 10 μm to about 400 μm, about 10 μm to about 350 μm, about10 μm to about 300 μm, about 10 μm to about 250 μm, about 10 μm to about200 μm, about 10 μm to about 150 μm, about 10 μm to about 100 μm, about10 μm to about 80 μm, about 10 μm to about 60 μm, about 10 μm to about40 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15μm to about 500 μm, about 15 μm to about 450 μm, about 15 μm to about400 μm, about 15 μm to about 350 μm, about 15 μm to about 300 μm, about15 μm to about 250 μm, about 15 μm to about 200 μm, about 15 μm to about150 μm, about 15 μm to about 100 μm, about 15 μm to about 80 μm, about15 μm to about 60 μm, about 15 μm to about 40 μm, about 15 μm to about20 μm, about 20 μm to about 500 μm, about 20 μm to about 450 μm, about20 μm to about 400 μm, about 20 μm to about 350 μm, about 20 μm to about300 μm, about 20 μm to about 250 μm, about 20 μm to about 200 μm, about20 μm to about 150 μm, about 20 μm to about 100 μm, about 20 μm to about80 μm, about 20 μm to about 60 μm, about 20 μm to about 40 μm, about 40μm to about 500 about 40 μm to about 450 μm, about 40 μm to about 400μm, about 40 μm to about 350 μm, about 40 μm to about 300 μm, about 40μm to about 250 μm, about 40 μm to about 200 μm, about 40 μm to about150 μm, about 40 μm to about 100 μm, about 40 μm to about 80 μm, about40 μm to about 60 μm, about 60 μm to about 500 about 60 μm to about 450μm, about 60 μm to about 400 μm, about 60 μm to about 350 μm, about 60μm to about 300 μm, about 60 μm to about 250 μm, about 60 μm to about200 μm, about 60 μm to about 150 μm, about 60 μm to about 100 μm, about60 μm to about 80 μm, about 80 μm to about 500 about 80 μm to about 450μm, about 80 μm to about 400 μm, about 80 μm to about 350 μm, about 80μm to about 300 μm, about 80 μm to about 250 μm, about 80 μm to about200 μm, about 80 μm to about 150 μm, about 80 μm to about 100 μm, about100 μm to about 500 about 100 μm to about 450 μm, about 100 μm to about400 μm, about 100 μm to about 350 μm, about 100 μm to about 300 μm,about 100 μm to about 250 μm, about 100 μm to about 200 μm, about 100 μmto about 150 μm, about 150 μm to about 500 about 150 μm to about 450 μm,about 150 μm to about 400 μm, about 150 μm to about 350 μm, about 150 μmto about 300 μm, about 150 μm to about 250 μm, about 150 μm to about 200μm, about 200 μm to about 500 about 200 μm to about 450 μm, about 200 μmto about 400 μm, about 200 μm to about 350 μm, about 200 μm to about 300μm, about 200 μm to about 250 μm, about 250 μm to about 500 about 250 μmto about 450 μm, about 250 μm to about 400 μm, about 250 μm to about 350μm, about 250 μm to about 300 μm, about 300 μm to about 500 about 300 μmto about 450 μm, about 300 μm to about 400 μm, about 300 μm to about 350μm, about 350 μm to about 500 about 350 μm to about 450 μm, about 350 μmto about 400 μm, about 400 μm to about 500 about 400 μm to about 450 μm,about 450 μm to about 500 μm.

In some embodiments, particles can be of a nanometer scale (e.g.,particles can have a diameter or maximum cross-sectional dimension ofabout 10 nm to about 900 nm, about 10 nm to about 850 nm, about 10 nm toabout 800 nm, about 10 nm to about 750 nm, about 10 nm to about 700 nm,about 10 nm to about 650 nm, about 10 nm to about 600 nm, about 10 nm toabout 550 nm, about 10 nm to about 500 nm, about 10 nm to about 450 nm,about 10 nm to about 400 nm, about 10 nm to about 350 nm, about 10 nm toabout 300 nm, about 10 nm to about 250 nm, about 10 nm to about 200 nm,about 10 nm to about 150 nm, about 10 nm to about 100 nm, about 10 nm toabout 50 nm, about 50 nm to about 900 nm, about 50 nm to about 850 nm,about 50 nm to about 800 nm, about 50 nm to about 750 nm, about 50 nm toabout 700 nm, about 50 nm to about 650 nm, about 50 nm to about 600 nm,about 50 nm to about 550 nm, about 50 nm to about 500 nm, about 50 nm toabout 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm,about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm toabout 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm,about 100 nm to about 900 nm, about 100 nm to about 850 nm, about 100 nmto about 800 nm, about 100 nm to about 750 nm, about 100 nm to about 700nm, about 100 nm to about 650 nm, about 100 nm to about 600 nm, about100 nm to about 550 nm, about 100 nm to about 500 nm, about 100 nm toabout 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350nm, about 100 nm to about 300 nm, about 100 nm to about 250 nm, about100 nm to about 200 nm, about 100 nm to about 150 nm, about 150 nm toabout 900 nm, about 150 nm to about 850 nm, about 150 nm to about 800nm, about 150 nm to about 750 nm, about 150 nm to about 700 nm, about150 nm to about 650 nm, about 150 nm to about 600 nm, about 150 nm toabout 550 nm, about 150 nm to about 500 nm, about 150 nm to about 450nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm toabout 200 nm, about 200 nm to about 900 nm, about 200 nm to about 850nm, about 200 nm to about 800 nm, about 200 nm to about 750 nm, about200 nm to about 700 nm, about 200 nm to about 650 nm, about 200 nm toabout 600 nm, about 200 nm to about 550 nm, about 200 nm to about 500nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about200 nm to about 350 nm, about 200 nm to about 300 nm, about 200 nm toabout 250 nm, about 250 nm to about 900 nm, about 250 nm to about 850nm, about 250 nm to about 800 nm, about 250 nm to about 750 nm, about250 nm to about 700 nm, about 250 nm to about 650 nm, about 250 nm toabout 600 nm, about 250 nm to about 550 nm, about 250 nm to about 500nm, about 250 nm to about 450 nm, about 250 nm to about 400 nm, about250 nm to about 350 nm, about 250 nm to about 300 nm, about 300 nm toabout 900 nm, about 300 nm to about 850 nm, about 300 nm to about 800nm, about 300 nm to about 750 nm, about 300 nm to about 700 nm, about300 nm to about 650 nm, about 300 nm to about 600 nm, about 300 nm toabout 550 nm, about 300 nm to about 500 nm, about 300 nm to about 450nm, about 300 nm to about 400 nm, about 300 nm to about 350 nm, about350 nm to about 900 nm, about 350 nm to about 850 nm, about 350 nm toabout 800 nm, about 350 nm to about 750 nm, about 350 nm to about 700nm, about 350 nm to about 650 nm, about 350 nm to about 600 nm, about350 nm to about 550 nm, about 350 nm to about 500 nm, about 350 nm toabout 450 nm, about 350 nm to about 400 nm, about 400 nm to about 900nm, about 400 nm to about 850 nm, about 400 nm to about 800 nm, about400 nm to about 750 nm, about 400 nm to about 700 nm, about 400 nm toabout 650 nm, about 400 nm to about 600 nm, about 400 nm to about 550nm, about 400 nm to about 500 nm, about 400 nm to about 450 nm, about450 nm to about 900 nm, about 450 nm to about 850 nm, about 450 nm toabout 800 nm, about 450 nm to about 750 nm, about 450 nm to about 700nm, about 450 nm to about 650 nm, about 450 nm to about 600 nm, about450 nm to about 550 nm, about 450 nm to about 500 nm, about 500 nm toabout 900 nm, about 500 nm to about 850 nm, about 500 nm to about 800nm, about 500 nm to about 750 nm, about 500 nm to about 700 nm, about500 nm to about 650 nm, about 500 nm to about 600 nm, about 500 nm toabout 550 nm, about 550 nm to about 900 nm, about 550 nm to about 850nm, about 550 nm to about 800 nm, about 550 nm to about 750 nm, about550 nm to about 700 nm, about 550 nm to about 650 nm, about 550 nm toabout 600 nm, about 600 nm to about 900 nm, about 600 nm to about 850nm, about 600 nm to about 800 nm, about 600 nm to about 750 nm, about600 nm to about 700 nm, about 600 nm to about 650 nm, about 650 nm toabout 900 nm, about 650 nm to about 850 nm, about 650 nm to about 800nm, about 650 nm to about 750 nm, about 650 nm to about 700 nm, about700 nm to about 900 nm, about 700 nm to about 850 nm, about 700 nm toabout 800 nm, about 700 nm to about 750 nm, about 750 nm to about 900nm, about 750 nm to about 850 nm, about 750 nm to about 800 nm, about800 nm to about 900 nm, about 800 nm to about 850 nm, or about 850 nm toabout 900 nm.

In some embodiments, a particle has a diameter or volume that is aboutthe diameter of a single cell (e.g., a single cell under evaluation).

In some embodiments, the diffusion-restricted nuclease is covalentlyconjugated to a polymer. The polymer can be a natural polymer, asynthetic polymer, or a combination of both natural and syntheticpolymers. Examples of natural polymers include, without limitation,proteins, sugars such as deoxyribonucleic acid, rubber, cellulose,starch (e.g., amylose and amylopectin), polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers include,without limitation, acrylics, nylons, silicones, spandex, viscose rayon,polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate,polyethylene glycol, polyurethanes, polylactic acid, silica,polystyrene, polyacrylonitrile, polybutadiene, polycarbonate,polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene),poly(ethylene oxide), poly(ethylene terephthalate), polyethylene,polyisobutylene, poly(methyl methacrylate), poly(oxymethylene),polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene),poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride),poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinylfluoride), and/or combinations (e.g., co-polymers) thereof.

In some embodiments, the polymer can have an average molecular weight ofat least about 100 Da (dalton), at least about 200 Da, at least about300 Da, at least about 400 Da, at least about 500 Da, at least about 600Da, at least about 700 Da, at least about 800 Da, at least about 900 Da,at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, atleast about 4 kDa, at least about 5 kDa, at least about 6 kDa, at leastabout 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about10 kDa, at least about 20 kDa, at least about 50 kDa, or at least about100 kDa. In some embodiments, the polymer can have a molecular weightabout 100 Da to about 500 Da, about 500 Da to about 1 kDa, about 1 kDato about 5 kDa, about 5 kDa to about 20 kDa, about 20 kDa to about 100kDa.

In some embodiments, the polymer can have a hydrodynamic radius nolarger than 100 μm (e.g., no larger than 95 μm, 90 μm, 85 μm, 80 μm, 75μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm).

In some embodiments, the diffusion-restricted nuclease is conjugated toa bead, a particle, or a polymer through non-covalent interactions,e.g., electrostatic interactions, hydrogen bonds, Van der Waalsinteractions, hydrophobic interactions, ion-induced dipole interactions,dipole-induced dipole interactions, or any combination thereof.

III. Contacting the Second Area of the Array with a Solution Comprisinga Diffusion-Restricted Nuclease

In some embodiments, the solution comprising a diffusion-restrictednuclease is added automatically (e.g., by a robot) or manually (e.g., bypipetting). In some embodiments, the solution comprising adiffusion-restricted nuclease is added dropwise by a pipette. In someembodiments, the solution comprising a diffusion-restricted nuclease isadded to contact all or a portion of the second area of the array. Insome embodiments, the solution comprising a diffusion-restrictednuclease is added to all or a portion of a surface of thenon-permeabilized biological sample that is not facing or contacting thearray.

In some embodiments, the solution is added vertically above the secondarea of the array. In some embodiments, the solution is present inliquid form, such that the second area is covered by the solution. Insome alternative embodiments, the diffusion-restricted nuclease iscontacted to the second area in a gel form or a semifluidic form.

In some embodiments, the concentration of the diffusion-restrictednuclease in the solution is at least about 0.0001 mg/mL, at least about0.001 mg/mL, at least about 0.01 mg/mL, at least about 0.02 mg/mL, atleast about 0.03 mg/mL, at least about 0.04 mg/mL, at least about 0.05mg/mL, at least about 0.06 mg/mL, at least about 0.07 mg/mL, at leastabout 0.08 mg/mL, at least about 0.09 mg/mL, at least about 0.1 mg/mL,at least about 0.2 mg/mL, at least about 0.3 mg/mL, at least about 0.4mg/mL, at least about 0.5 mg/mL, at least about 0.6 mg/mL, at leastabout 0.7 mg/mL, at least about 0.8 mg/mL, at least about 0.9 mg/mL, atleast about 1 mg/mL, at least about 2 mg/mL, at least about 3 mg/mL, atleast about 4 mg/mL, at least about 5 mg/mL, at least about 6 mg/mL, atleast about 7 mg/mL, at least about 8 mg/mL, at least about 9 mg/mL, atleast about 10 mg/mL, at least about 20 mg/mL, at least about 30 mg/mL,at least about 40 mg/mL, at least about 50 mg/mL, at least about 60mg/mL, at least about 70 mg/mL, at least about 80 mg/mL, at least about90 mg/mL, at least about 100 mg/mL, at least about 200 mg/mL, at leastabout 500 mg/maL, at least about 800 mg/mL, or at least about 1000mg/mL.

In some embodiments, the concentration of the diffusion-restrictednuclease in the solution is at least about 0.001 units/mL, at leastabout 0.01 units/mL, at least about 0.02 units/mL, at least about 0.03units/mL, at least about 0.04 units/mL, at least about 0.05 units/mL, atleast about 0.06 units/mL, at least about 0.07 units/mL, at least about0.08 units/mL, at least about 0.09 units/mL, at least about 0.1units/mL, at least about 0.2 units/mL, at least about 0.3 units/mL, atleast about 0.4 units/mL, at least about 0.5 units/mL, at least about0.6 units/mL, at least about 0.7 units/mL, at least about 0.8 units/mL,at least about 0.9 units/mL, at least about 1 unit/mL, at least about 2units/mL, at least about 3 units/mL, at least about 4 units/mL, at leastabout 5 units/mL, at least about 6 units/mL, at least about 7 units/mL,at least about 8 units/mL, at least about 9 units/mL, at least about 10units/mL, at least about 20 units/mL, at least about 30 units/mL, atleast about 40 units/mL, at least about 50 units/mL, at least about 60units/mL, at least about 70 units/mL, at least about 80 units/mL, atleast about 90 units/mL, or at least about 100 units/mL.

In some embodiments, the second area of the array can be contacted bythe solution for at least about 5 minutes, at least about 10 minutes, atleast about 15 minutes, at least about 20 minutes, at least about 25minutes, at least about 30 minutes, at least about 40 minutes, at leastabout 50 minutes, or at least about 1 hour, or greater at a temperatureof about 4° C., about 10° C., about 20° C., about 25° C., about 30° C.,about 32° C., about 34° C., about 35° C., about 36° C., about 37° C.,about 38° C., about 39° C., about 40° C., about 45° C., or about 50° C.

In some embodiments, the second area of the array can be contacted bythe solution for about 5 minutes to about 1 hour, about 5 minutes toabout 50 minutes, about 5 minutes to about 40 minutes, about 5 minutesto about 30 minutes, about 5 minutes to about 20 minutes, about 5minutes to about 10 minutes, about 10 minutes to about 1 hour, about 10minutes to about 50 minutes, about 10 minutes to about 40 minutes, about10 minutes to about 30 minutes, about 10 minutes to about 20 minutes,about 20 minutes to about 1 hour, about 20 minutes to about 50 minutes,about 20 minutes to about 40 minutes, about 20 minutes to about 30minutes, about 30 minutes to about 1 hour, about 30 minutes to about 50minutes, about 30 minutes to about 40 minutes, about 40 minutes to about1 hour, about 40 minutes to about 50 minutes, or about 50 minutes toabout 1 hour, at a temperature of about 4° C. to about 35° C., about 4°C. to about 30° C., about 4° C. to about 25° C., about 4° C. to about20° C., about 4° C. to about 15° C., about 4° C. to about 10° C., about10° C. to about 35° C., about 10° C. to about 30° C., about 10° C. toabout 25° C., about 10° C. to about 20° C., about 10° C. to about 15°C., about 15° C. to about 35° C., about 15° C. to about 30° C., about15° C. to about 25° C., about 15° C. to about 20° C., about 20° C. toabout 35° C., about 20° C. to about 30° C., about 20° C. to about 25°C., about 25° C. to about 35° C., about 25° C. to about 30° C., or about30° C. to about 35° C.

IV. Removing the Diffusion-Restricted Nuclease from the Second Area ofthe Array

In some embodiments, the solution comprising a diffusion-restrictednuclease is removed by pipetting. In some embodiments, thediffusion-restricted nuclease is removed by wicking (e.g., by anabsorption paper). In some embodiments, the diffusion-restrictednuclease is removed by washing (e.g., using a wash buffer). In someembodiments, the wash buffer can be added to contact the second area ofthe array then removed by pipetting, wicking, or other methods known inthe art. In some embodiments, a combination of removing methods can beused. In some embodiments, contacting and removing steps can be repeated(e.g., at least 2 times, 3 times, 4 times, or greater). In someembodiments, a drying step can be performed after washing (e.g., airdry).

In some embodiments, the wash buffer is added automatically (e.g., by arobot) or manually (e.g., by pipetting). In some embodiments, the washbuffer is added vertically above the second area of the array. In someembodiments, the wash buffer is added dropwise by a pipette. In someembodiments, the wash buffer is added to contact all or a portion of thesecond area of the array. In some embodiments, the wash buffer is addedto all or a portion of a surface of the non-permeabilized biologicalsample that is not facing or contacting the array.

In some embodiments, the wash buffer comprises a nuclease inhibitor soas to stop the diffusion-restricted nuclease from functioning. In someembodiments, the washing buffer is 1× TE buffer, 1×TAE buffer, 1×TBEbuffer, or PBS. In some embodiments, the wash buffer contains a buffer(e.g., Tris, MOPS, HEPES, MES, or any other buffer known in the art),chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)) and/ormetal ions (e.g., Mg²⁺). In some embodiments, the wash buffer can have apH that is about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0, or about5.0 to 5.5, about 5.5 to 6.0, about 6.0 to 6.5, about 6.5 to 7.0, about7.0 to 7.5, about 7.5 to 8.0, about 8.0 to 8.5, about 8.5 to 9.0, about9.0 to 9.5, or about 9.5 to 10.0.

In some embodiments, the second area of the array is contacted by thewash buffer for about 5 seconds to about 1 hour, about 5 seconds toabout 50 minutes, about 5 seconds to about 40 minutes, about 5 secondsto about 30 minutes, about 5 seconds to about 20 minutes, about 5seconds to about 10 minutes, about 5 seconds to about 5 minutes, about 5seconds to about 1 minute, about 5 seconds to about 30 seconds, about 5seconds to about 10 seconds, about 10 seconds to about 1 hour, about 10seconds to about 50 minutes, about 10 seconds to about 40 minutes, about10 seconds to about 30 minutes, about 10 seconds to about 20 minutes,about 10 seconds to about 10 minutes, about 10 seconds to about 5minutes, about 10 seconds to about 1 minute, about 10 seconds to about30 seconds, about 30 seconds to about 1 hour, about 30 seconds to about50 minutes, about 30 seconds to about 40 minutes, about 30 seconds toabout 30 minutes, about 30 seconds to about 20 minutes, about 30 secondsto about 10 minutes, about 30 seconds to about 5 minutes, about 30seconds to about 1 minute, about 1 minute to about 1 hour, about 1minute to about 50 minutes, about 1 minute to about 40 minutes, about 1minute to about 30 minutes, about 1 minute to about 20 minutes, about 1minute to about 10 minutes, about 1 minute to about 5 minutes, about 5minutes to about 1 hour, about 5 minutes to about 50 minutes, about 5minutes to about 40 minutes, about 5 minutes to about 30 minutes, about5 minutes to about 20 minutes, about 5 minutes to about 10 minutes,about 10 minutes to about 1 hour, about 10 minutes to about 50 minutes,about 10 minutes to about 40 minutes, about 10 minutes to about 30minutes, about 10 minutes to about 20 minutes, about 20 minutes to about1 hour, about 20 minutes to about 50 minutes, about 20 minutes to about40 minutes, about 20 minutes to about 30 minutes, about 30 minutes toabout 1 hour, about 30 minutes to about 50 minutes, about 30 minutes toabout 40 minutes, about 40 minutes to about 1 hour, about 40 minutes toabout 50 minutes, or about 50 minutes to about 1 hour at a temperatureof about 4° C. to about 35° C., about 4° C. to about 30° C., about 4° C.to about 25° C., about 4° C. to about 20° C., about 4° C. to about 15°C., about 4° C. to about 10° C., about 10° C. to about 35° C. to about10° C. to about 30° C., about 10° C. to about 25° C., about 10° C. toabout 20° C., about 10° C. to about 15° C., about 15° C. to about 35°C., about 15° C. to about 30° C., about 15° C. to about 25° C., about15° C. to about 20° C., about 20° C. to about 35° C., about 20° C. toabout 30° C., about 20° to about 25° C., about 25° C. to about 35° C.,about 25° C. to about 30° C., or about 30° C. to about 35° C.

In some embodiments, the second area of the array can be contacted bythe wash buffer for at least about 1 second, at least about 5 seconds,at least about 10 seconds, at least about 15 seconds, at least about 20seconds, at least about 30 seconds, at least about 45 seconds, at leastabout 1 minutes, at least about 5 minutes, at least about 10 minutes, atleast about 15 minutes, at least about 20 minutes, at least about 25minutes, at least about 30 minutes, at least about 40 minutes, at leastabout 50 minutes, at least about 1 hour, at a temperature of about 4°C., about 10° C., about 20° C., about 25° C., about 30° C., about 32°C., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., about 40° C., about 45° C. or about 50° C.

In some embodiments, the solution comprising the diffusion-restrictednuclease contains a gel precursor material (e.g., polyacrylamide) andthe diffusion-restricted nuclease is removed by first adding a solutioncomprising a cross-linking agent (e.g., APS/TEMED) to polymerize or gelthe gel precursor material, followed by separating the solution presentas a gel from the second area of the array.

In some embodiments, the solution comprising the diffusion-restrictednuclease is present as a gel, and the solution can be removed byseparating the gel from the second area of the array.

In some embodiments, the solution comprising the diffusion-restrictednuclease is removed by reducing the temperature of the solution to nomore than about 0° C., no more than about −5° C., no more than about−10° C., no more than about −20° C., no more than about −50° C., no morethan about −80° C. (e.g., lower than the melting point of the solution),followed by separating the solution in solid form from the second areaof the array.

In some embodiments, the diffusion-restricted nuclease is linked to amagnetic bead (or a magnetic particle, or other magnetic substancethereof) and the diffusion-restricted nuclease can be removed byapplying a magnetic field.

V. Methods for Determining a Location of a Target Nucleic Acid

Provided herein are methods for determining a location of a targetnucleic acid in a biological sample disposed onto an array (e.g., any ofthe arrays described herein), where the array has a first area coveredby the biological sample (e.g., any of the first areas described herein)and a second area not covered by the biological sample (e.g., any of thesecond areas described herein); where the array comprises a plurality ofcapture probes (e.g., any of the capture probes described herein), wherea capture probe of the plurality of capture probes comprises a spatialbarcode (e.g., any of the spatial barcodes described herein) and acapture domain (e.g., any of the capture domains as described herein),where the methods include: (a) contacting the second area of the arraywith a solution comprising a diffusion-restricted nuclease (e.g., any ofthe diffusion-restricted nucleases described herein); (b) removing thediffusion-restricted nuclease from the second area of the array (e.g.,using any of the methods for removing the diffusion-restricted nucleasesdescribed herein); (c) permeabilizing the biological sample, such thatthe capture domain binds to the target nucleic acid (e.g., using any ofthe methods for permeabilizing a biological sample described herein);and (d) determining (i) all or a portion of the sequence of the spatialbarcode, or a complement thereof, and (ii) all or a portion of thesequence of the target nucleic acid, or a complement thereof, and usingthe sequences of (i) and (ii) to determine the location of the targetnucleic acid in the biological sample.

Non-limiting examples of a target nucleic acid include DNA analytes suchas genomic DNA, methylated DNA, specific methylated DNA sequences,fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, andRNA/DNA hybrids.

Non-limiting examples of the target nucleic acid also include RNAanalytes such as various types of coding and non-coding RNA. Examples ofthe different types of RNA analytes include messenger RNA (mRNA),ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viralRNA. The RNA can be a transcript (e.g., present in a tissue section).The RNA can be small (e.g., less than 200 nucleic acid bases in length)or large (e.g., RNA greater than 200 nucleic acid bases in length).Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transferRNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), smallnucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derivedsmall RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can bedouble-stranded RNA or single-stranded RNA. The RNA can be circular RNA.The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

In some embodiments, the target nucleic acid can include at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10 disease-causing mutations (e.g.,cancer-causing mutations). In some embodiments, the target nucleic acidincludes polymorphism, gene amplification, or chromosomal translocation.

In some embodiments, the biological sample as described herein can bestained or imaged by techniques known in the art. In some embodiments,the staining includes optical labels as described herein, including, butnot limited to, fluorescent, radioactive, chemiluminescent,calorimetric, or colorimetric detectable labels. In some embodiments,the staining includes a fluorescent antibody directed to a targetanalyte (e.g., cell surface or intracellular proteins) in the biologicalsample. In some embodiments, the staining includes animmunohistochemistry stain directed to a target analyte (e.g., cellsurface or intracellular proteins) in the biological sample. In someembodiments, the staining includes a chemical stain such as hematoxylinand eosin (H&E) or periodic acid-schiff (PAS). In some embodiments,significant time (e.g., days, months, or years) can elapse betweenstaining and/or imaging the biological sample and performing analysis.

VI. Methods for Determining a Location of a Target Analyte

Provided herein are methods for determining a location of a targetanalyte in a biological sample that include: (a) contacting a pluralityof analyte capture agents (e.g., any of the analyte capture agentsdescribed herein) to the biological sample, where an analyte captureagent of the plurality of analyte capture agents comprises an analytebinding moiety barcode (e.g., any of the analyte binding moiety barcodesdescribed herein), an analyte capture sequence (e.g., any of the analytecapture sequences described herein), and an analyte binding moiety(e.g., any of the analyte binding moieties described herein) that bindsspecifically to the target analyte; (b) disposing the biological sampleonto an array (e.g., any of the arrays described herein), where thearray has a first area (e.g., any of the first areas described herein)covered by the biological sample and a second area (e.g., any of thesecond areas described herein) not covered by the biological sample,where the array comprises a plurality of capture probes, where a captureprobe of the plurality comprises a spatial barcode (e.g., any of thespatial barcodes described herein) and a capture domain (e.g., any ofthe capture domains described herein) that binds specifically to theanalyte capture sequence; (c) contacting the second area of the arraywith a solution comprising a diffusion-restricted nuclease (e.g., any ofthe diffusion-restricted nucleases described herein); (d) removing thediffusion-restricted nuclease from the second area of the array (e.g.,using any of the methods for removing the diffusion-restricted nucleasesdescribed herein); and (e) determining (i) all or a portion of thesequence of the spatial barcode, or a complement thereof, and (ii) allor a portion of the sequence of the analyte binding moiety barcode, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target analyte in the biological sample.

VII. Methods for Decreasing the Background binding of a Target NucleicAcid or Target Analyte

Provided herein are methods for decreasing background binding of atarget nucleic acid on an array that include: (a) disposing a biologicalsample onto an array (e.g., any of the arrays described herein), whereinthe array has a first area (e.g., any of the first areas describedherein) covered by the biological sample and a second area (e.g., any ofthe second areas described herein) not covered by the biological sample,wherein the array comprises a plurality of capture probes (e.g., any ofthe capture probes described herein). wherein a capture probe of theplurality of capture probes comprises a spatial barcode (e.g., any ofthe spatial barcodes described herein) and a capture domain (e.g., anyof the capture domains described herein); (b) contacting the second areaof the array with a solution comprising a diffusion-restricted nuclease(e.g., any of the diffusion-restricted nucleases described herein), (c)removing the diffusion-restricted nuclease from the second area of thearray (e.g., using any of the methods for removing thediffusion-restricted nucleases described herein); and (d) permeabilizingthe biological sample (e.g., using any of the methods for permeabilizinga biological sample described herein), such that the capture domainbinds to the target nucleic acid in the first area, thereby decreasingthe background binding of a target nucleic acid on the array. Someembodiments further include: (e) (e) determining (i) all or a portion ofthe sequence of the spatial barcode, or a complement thereof, and (ii)all or a portion of the sequence of the target nucleic acid, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target nucleic acid in the biological sample.

Provided herein are methods for decreasing background binding of atarget analyte on an array that include: (a) contacting a plurality ofanalyte capture agents (e.g., any of the analyte capture agentsdescribed herein) to the biological sample, wherein an analyte captureagent of the plurality of analyte capture agents comprises an analytebinding moiety barcode (e.g., any of the analyte binding moiety barcodesdescribed herein), an analyte capture sequence (e.g., any of the analytecapture sequences described herein), and an analyte binding moiety(e.g., any of the analyte binding moieties described herein) that bindsspecifically to the target analyte; (b) disposing the biological sampleonto an array (e.g., any of the arrays described herein), wherein thearray has a first area (e.g., any of the first areas described herein)covered by the biological sample and a second area (e.g., any of thesecond areas described herein) not covered by the biological sample,wherein the array comprises a plurality of capture probes, wherein acapture probe of the plurality comprises a spatial barcode (e.g., any ofthe spatial barcodes described herein) and a capture domain (e.g., anyof the capture domains described herein) that binds specifically to theanalyte capture sequence; (c) contacting the second area of the arraywith a solution comprising a diffusion-restricted nuclease (e.g., any ofthe diffusion-restricted nucleases described herein); (d) removing thediffusion-restricted nuclease from the second area of the array (e.g.,using any of the methods for removing the diffusion-restricted nucleasesdescribed herein), and (e) permeabilizing the biological sample (e.g.,using any of the methods for permeabilizing a biological sampledescribed herein), such that the capture domain binds to the analytecapture sequence in the first area, thereby decreasing the backgroundbinding of a target analyte on the array. In some embodiments, themethods further include: (f) determining (i) all or a portion of thesequence of the spatial barcode, or a complement thereof, and (ii) allor a portion of the sequence of the analyte binding moiety barcode, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target analyte in the biological sample. In someembodiments of these methods, step (a) is performed before step (b). Insome embodiments of these methods, step (b) is performed before step(a).

Provided herein are methods for decreasing the background binding of atarget analyte on a spatial array. For example, as seen in FIGS. 1 and2, the tissue sections are surrounded by, in these figures, backgroundfluorescence. One cause of the background fluorescence could be a resultof target analytes passively diffusing from the tissues and binding tothe capture domains of the capture probes that are around the tissue(e.g., a second area of the array). The methods provided herein providefor the digestion, complete or partial, of the nucleic acid captureprobes that are not under the biological sample (e.g., the first area).It is contemplated that by completely or partially rendering the captureprobe domains adjacent to the biological sample inoperable (e.g., viadigestion with an endonuclease or exonuclease, or both), binding oftarget analytes will be minimized or eliminated, thereby decreasingbackground on the spatial array. As such, methods for decreasingbackground on a spatial array comprise a biological sample disposed on aspatial array, wherein the spatial array has a first area covered by thebiological sample and a second area not covered by the biologicalsample, wherein the spatial array comprises a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises a spatial barcode and a capture domain. The second area of thearray is covered with a solution comprising a diffusion-restrictednuclease (any of the nucleases as described herein restricted by anymeans described herein), which is allowed to be in contact with thesecond area for a period of time to completely or partially digest allor a portion of the capture probe domain, after which point the nucleaseis removed for the spatial array, for example by washing. Followingnuclease treatment of the second area of the spatial array, it iscontemplated that minimal binding of target analytes from the biologicalsample will occur. The biological sample can be permeabilized therebyreleasing the target analytes from the tissues, target analytes whichcan bind to the capture domains in the first are and not the second areaof the spatial array. The bound analytes can complete the spatialtranscriptomics workflow, for example by extension of the capture probeto create complementary sequences to the spatial barcode and (at least)all or a portion of the target analyte sequence, sequences which can bedetermined to identify the target analyte and its location in thebiological sample.

VIII. Kits

Also provided herein are kits that include: an array comprising aplurality of capture probes (e.g., any of the exemplary capture probesdescribed herein), where a capture probe of the plurality of captureprobes comprises a spatial barcode (e.g., any of the exemplary spatialbarcodes described herein) and a capture domain (e.g., any of theexemplary capture domains described herein); and a diffusion-restrictednuclease (e.g., any of the exemplary diffusion-restricted nucleasesdescribed herein).

Also provided herein are kits that include: a plurality of analytecapture agents (e.g., any of the exemplary analyte capture agentsdescribed herein), where an analyte capture agent of the plurality ofanalyte capture agents comprises an analyte binding moiety barcode(e.g., any of the exemplary analyte binding moiety barcodes describedherein), an analyte capture sequence (e.g., any of the exemplary analytecapture sequences described herein), and an analyte binding moiety(e.g., any of the exemplary analyte binding moieties described herein)that binds specifically to a target analyte; an array comprising aspatial barcode (e.g., any of the exemplary spatial barcodes describedherein) and a capture domain (e.g., any of the exemplary capture domainsdescribed herein) that binds specifically to the analyte capturesequence; and a diffusion-restricted nuclease (e.g., any of theexemplary diffusion-restricted nucleases described herein).

In some embodiments of any of the kits described herein, thediffusion-restricted nuclease degrades single-stranded nucleic acids. Insome embodiments of any of the kits described herein, thediffusion-restricted nuclease degrades double-stranded nucleic acids. Insome embodiments of any of the kits described herein, thediffusion-restricted nuclease is covalently linked to a bead (e.g., anyof the exemplary beads described herein). In some embodiments of any ofthe kits described herein, the diffusion-restricted nuclease iscovalently linked to a particle (e.g., any of the exemplary particlesdescribed herein). In some embodiments of any of the kits describedherein, the diffusion-restricted nuclease is covalently linked to apolymer (e.g., any of the exemplary polymers described herein, such as apolyethylene glycol). In some embodiments of any of the kits describedherein, the diffusion-restricted nuclease is an endonuclease. In someembodiments of any of the kits described herein, thediffusion-restricted nuclease is an exonuclease (e.g., a 3′ to 5′exonuclease). In some embodiments of any of the kits described herein,the exonuclease is a DNAse. In some embodiments of any of the kitsdescribed herein, the array comprises a slide or a bead array.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for determining a location of a target nucleic acid in abiological sample disposed onto an array, wherein the array has a firstarea covered by the biological sample and a second area not covered bythe biological sample, wherein the array comprises a plurality ofcapture probes, wherein a capture probe of the plurality of captureprobes comprises a spatial barcode and a capture domain, the methodcomprising: (a) contacting the second area of the array with a solutioncomprising a diffusion-restricted nuclease; (b) removing thediffusion-restricted nuclease from the second area of the array; (c)permeabilizing the biological sample, such that the capture domain bindsto the target nucleic acid; and (d) determining (i) all or a portion ofthe sequence of the spatial barcode, or a complement thereof, and (ii)all or a portion of the sequence of the target nucleic acid, or acomplement thereof, and using the sequences of (i) and (ii) to determinethe location of the target nucleic acid in the biological sample.
 2. Themethod of claim 1, wherein the diffusion-restricted nuclease degradessingle-stranded or double-stranded nucleic acids.
 3. (canceled)
 4. Themethod of claim 1, wherein the diffusion-restricted nuclease iscovalently linked to a bead, a particle, or a polymer.
 5. (canceled) 6.The method of claim 1, wherein the diffusion-restricted nuclease is anendonuclease or an exonuclease. 7.-8. (canceled)
 9. The method of claim1, wherein the removing in step (b) comprises washing.
 10. The method ofclaim 1, wherein the array comprises a slide or a bead.
 11. (canceled)12. The method of claim 1, wherein the determining in step (d) comprisessequencing (i) all or a portion of the sequence of the spatial barcode,or a complement thereof, and (ii) all or a portion of the sequence ofthe target nucleic acid, or a complement thereof.
 13. (canceled)
 14. Themethod of claim 1, wherein the determining in step (d) comprisesextending the capture probe using the target nucleic acid as thetemplate.
 15. The method of claim 1, wherein the biological sample is atissue section. 16.-35. (canceled)
 36. A method for decreasingbackground binding of a target nucleic acid on an array, comprising: (a)disposing a biological sample onto an array, wherein the array has afirst area covered by the biological sample and a second area notcovered by the biological sample, wherein the array comprises aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain; (b)contacting the second area of the array with a solution comprising adiffusion-restricted nuclease, (c) removing the diffusion-restrictednuclease from the second area of the array; and (d) permeabilizing thebiological sample, such that the capture domain binds to the targetnucleic acid in the first area, thereby decreasing the backgroundbinding of a target nucleic acid on the array.
 37. The method of claim36, further comprising determining (i) all or a portion of the sequenceof the spatial barcode, or a complement thereof, and (ii) all or aportion of the sequence of the target nucleic acid, or a complementthereof, and using the sequences of (i) and (ii) to determine thelocation of the target nucleic acid in the biological sample.
 38. Themethod of claim 36, wherein the diffusion-restricted nuclease degradessingle-stranded or double-stranded nucleic acids.
 39. (canceled)
 40. Themethod of claim 36, wherein the diffusion-restricted nuclease iscovalently linked to a bead, a particle, or a polymer.
 41. (canceled)42. The method of claim 36, wherein the diffusion-restricted nuclease isan endonuclease or an exonuclease. 43.-44. (canceled)
 45. The method ofclaim 36, wherein the removing comprises washing.
 46. The method ofclaim 36, wherein the array comprises a slide or a bead.
 47. (canceled)48. The method of claim 37, wherein the determining comprises sequencing(i) all or a portion of the sequence of the spatial barcode, or acomplement thereof, and (ii) all or a portion of the sequence of thetarget nucleic acid, or a complement thereof.
 49. (canceled)
 50. Themethod of claim 36, wherein the determining comprises extending thecapture probe using the target nucleic acid as the template.
 51. Themethod of claim 36, wherein steps (a) and (b) are performed atsubstantially the same time.
 52. A kit comprising: an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain; and adiffusion-restricted nuclease.
 53. (canceled)